It has been known generally that total bile acid concentration is elevated in patients with hepatobiliary diseases. The primary bile acids, cholic and chenodeoxycholic, are secreted by the liver as the glycine or taurine conjugates and stored in the gall bladder. Although some are absorbed from the gall bladder into the blood, most are secreted with the bile through the common bile duct into the lumen of the duodenum where they serve to facilitate the absorption of cholesterol and the digestion and absorption of fatty acids. The unused conjugated bile acids are absorbed into the blood vessels perfusing the duodenum and returned to the liver through the hepatic-portal system. Those bile acids not recovered in the duodenum are frequently converted to secondary bile acids by the action of intestinal flora. These too are absorbed into the blood and returned to the liver via the hepatic-portal system. In normal individuals the conjugated bile acids are removed from the circulation by the liver and recycled as conjugated primary bile acids. When the hepatocytes have been damaged by infection or chemicals, they are unable to recycle the bile acids in the usual manner. The quantity of affected cells is reflected by the quantitative relationship between the several primary bile acid conjugates and their corresponding secondary conjugates.
Numerous methodologies have been applied to the measurement of bile acids with varying degrees of success. Gas-liquid chromatography (G-LC) has been one of the most successful but suffers the disadvantages of requiring relatively large samples of patient serum, expensive equipment, and considerable sample pretreatment to isolate the conjugated from the unconjugated bile acids. Sample pretreatment includes isolation, saponification and derivation, rendering G-LC methods beyond the scope of ordinary laboratories.
A sensitive radioimmunoassay (RIA) for conjugates of cholic acid, a primary bile acid, was first described by Simmonds, et al., Radioimmunoassay of Conjugated Cholyl Bile Acids in Serum; Gastroenterology 65: 705-711 (1973). This Mayo Clinic group found their RIA to be specific for conjugates of cholic acid. Further clinical studies at the Mayo Clinic of Rochester, Minn., as reported by Korman, et al., in the New England Journal of Medicine, 290:1399 (1974), revealed elevated cholate conjugates in the sera of patients with chronic hepatitis in whom other liver function tests were normal and in patients with anticteric viral hepatitis. The clinical utility of this test was further demonstrated in 38 patients with chronic active hepatitis in whom the RIA of cholate conjugates proved to be a more sensitive indicator of disease than the traditional tests (bromosulfophthalein retention, prothrombin time, serum bilirubin, total proteins, alkaline phosphatase, and transaminase). Serum levels of bile acids, seen as the cholate conjugates, preceded serum enzyme elevations in those patients who ultimately relapsed. Although this method of the Mayo Clinic group showed good specifically for cholate conjugates, it could not distinguish between the glycine and the taurine cholate conjugates. G. M. Murphy, et al., The Preparation and Properties of an Antiserum for the Radioimmunoassay of Serum Conjugated Cholic Acid, Clinica Chimica Acta. 54: 81-89, (1974), repeated the work of Simmonds, et al., using a different method of hapten coupling and found essentially the same results.
While bile acids such as sulfolithocholic, cholic, chenodeoxycholic, deoxycholic, lithocholic and their conjugates with amino acids such as glycine and taurine can be prepared as the tritiated (.sup.3 H) forms, such as employed in the assays described above, these are undesirable as tracers in commercial immunoassays since they require: (1) the use of expensive beta counters, which most clinical laboratories do not have; (2) the use of scintillation fluids; (3) the use of special counting vials to which the tracer solution must be transferred; and (4) disposal of radioactive organic solvent waste.
Therefore, there is a need for a test that can be run directly on a serum sample yielding hepatic information about a specific bile acid conjugate and which can be used directly in an analytical instrument without special solution or vials.